Detection of binding reactions using labels detected by mediated catalytic electrochemistry

ABSTRACT

A method of detecting binding interactions and target molecules, such as proteins, protein fragments, recombinant proteins, recombinant protein fragments, extracellular matrix proteins, ligands, carbohydrates, steroids, hormones, drugs, drug candidates, immunoglobulins and receptors of eukaryotic, prokaryotic or viral origin, by mediated electrochemistry using labels that react with transition metal mediator complexes in a detectable catalytic redox reaction. These labels are attached directly to binders, target molecules, surrogate target molecules, or to affinity ligands capable of binding to the target or to surrogate target molecules capable of competing with the target for binding to another binder. The labels can be naturally present (endogenous) in the binder, target or affinity ligand, or constructed by the covalent attachment of the label to the binder, target, affinity ligand or surrogate target (exogenous).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part of application Ser. No.09/603,217, filed Jun. 26, 2000, which is a divisional application ofapplication Ser. No. 09/179,665, filed Oct. 27, 1998, now U.S. Pat. No.6,132,971, which is a divisional application of application Ser. No.08/667,338, filed Jun. 20, 1996, now U.S. Pat. No. 5,871,918, which is acontinuation-in-part of application Ser. No. 08/495,817, filed Jun. 27,1995; and a continuation-in-part of application Ser. No. 09/267,552filed Mar. 12, 1999, which is a continuation-in-part of application Ser.No. 08/667,338, filed Jun. 20, 1996, now U.S. Pat. No. 5,871,918, whichis a continuation-in-part of application Ser. No. 08/495,817, filed Jun.27, 1995; each of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to the detection of biologicalsubstances through binding interactions and, in particular, to methodsof detecting proteins and other substances by mediated catalyticelectrochemistry.

[0004] 2. Description of the Related Art

[0005] For many reasons, researchers are interested in the detection ofbiological substances such as nucleic acids, proteins, andcarbohydrates. Detection of such biomolecules can allow foridentification and development of targets for drug discovery and geneexpression analysis. The electrochemical detection of nucleic acidsprovides an alternative to fluorescent and radiochemical detectiontechniques that potentially eliminates the need for labeling.

[0006] The parent applications of the instant application, whose entirespecifications, drawings, and claims are specifically incorporatedherein by reference, disclose, among other inventions, sequencing andmethods of qualitatively and quantitatively detecting nucleic acidhybridization. Such inventions represent a major advance in the art andprovide oxidation-reduction reactions that function in a catalyticmanner without the addition of an enzyme or fluorescent label. Thesecatalytic reactions are useful for determining the presence or absenceof nucleic acids and provide for extremely accurate testing ofbiological samples. More specifically, catalytic oxidation has beenfound to be useful for quantitative detection of preselected nucleicacid bases (U.S. Pat. No. 5,871,918). The disclosures of each of thepatents and publications referred to herein are incorporated herein byreference.

[0007] The technology described in U.S. Pat. No. 5,871,918 utilizes thediscovery that nucleotide bases of DNA can be electrochemically oxidizedusing transition metal complexes as mediators. In this system, thenucleotide bases function as an array of endogenous redox-active labelsthat allow for ultrasensitive detection of DNA in conjunction withmicroelectrode methods. The detection reaction follows a two-stepmechanism involving reversible oxidation/reduction of the mediator.First the mediator is oxidized by an electrode. Then, the mediator isreduced by the preselected nucleotide base and reoxidized at theelectrode. In order for mediated oxidation of nucleic acids to proceedefficiently, the mediator and nucleotide base should have similaroxidation potentials. For example, catalytic oxidation of guanine can becarried out using the mediator, ruthenium²⁺ (2,2′bipyridine)₃ (Ru(bpy)₃²⁺). In solution, Ru(bpy)₃ ²⁺ exhibits a reversible redox couple at 1.05V (vs. Ag/AgCl reference), similar to the oxidation potential observedfor guanine (about 1.1 V vs. Ag/AgCl). Thus, addition ofguanine-containing DNA to a solution of Ru(bpy)₃ ²⁺ leads to catalyticenhancement in the electrochemical oxidation current via the followingreaction sequence:

Ru(bpy)₃ ²⁺→Ru(bpy)₃ ³⁺+e−

Ru(bpy)₃ ³⁺+DNA→DNA_(ox)+Ru(bpy)₃ ²⁺

[0008] where DNA_(ox) represents a DNA molecule in which guanine hasundergone a one electron oxidation.

[0009] The regeneration of reduced Ru(bpy)₃ ²⁺ by reaction with guaninecreates a catalytic cycle in which the presence of DNA is detected bytransfer of electrons from the preselected base to the electrode. Thenumber of turnovers obtained in the catalytic cycle depends on thenumber of electrons in the preselected base that can be oxidized by themediator and the number of preselected bases. In the case of guanineoxidation, Ru(bpy)₃ ²⁺ is capable of oxidizing guanine by at least twoelectrons (Armistead, P. M. and Thorp, H. H., Anal. Chem. 2000, 72,3764), and some reports suggest as many as 30 electrons obtained fromguanine through overoxidation steps (Thorp, H. H., Trends Biotechnol.1998, 16, 117). A typical DNA molecule will contain on average about oneguanine every four bases so even a small oligonucleotide will havemultiple guanines available for catalytic turnover of Ru(bpy)₃ ²⁺. As aresult of these properties, detection of nucleic acids via mediatedcatalytic electrochemistry is an extremely sensitive method.

[0010] Thus, in one embodiment of the prior invention, a nucleic acidsample is contacted with an oligonucleotide probe, which possesses asequence, at least a portion of which is capable of binding to a knownportion of the sequence in the nucleic acid sample, to form a hybridizednucleic acid, after which the hybridized nucleic acid is reacted with asuitable mediator, which is capable of oxidizing a preselected nucleicacid base in the hybridized nucleic acid sample in anoxidation-reduction reaction.

[0011] The selection of mediator in this prior work is dependent uponthe particular preselected nucleotide base chosen, and is readilydeterminable by those skilled in the art. Particularly preferredmediators include transition metal complexes that are capable ofparticipating in electron transfer with the preselected base such thatthe reduced form of the metal complex is regenerated, completing acatalytic cycle. An example of a suitable transition metal complex isRu(bpy)₃ ²⁺; however, the mediator or oxidizing agent may be anymolecule such as a cationic, anionic, non-ionic, or zwitterionicmolecule that is reactive with the preselected base at a uniqueoxidation potential to transfer electrons from the nucleic acid to theelectrode. All that is required is that the mediator be reacted with thehybridized nucleic acid sample under conditions sufficient to achievethe selective oxidation of the preselected base.

[0012] The oxidation-reduction rate is detected, for example, with adetection electrode, and the electronic signal may be detected by cyclicvoltammetry or other means known in the art. Hybridized DNA targetcontains guanine and is therefore more redox-active than the probestrand, which preferably is either selected or designed to contain aminimal number of guanines.

[0013] In U.S. Pat. No. 5,968,745 of Thorp et al., a polymer-electrodeis provided that is useful for the electrochemical detection of apreselected base in a nucleic acid. The polymer-electrode comprises: (a)a substrate having a conductive working surface; and (b) a polymer layeron the conductive working surface. The polymer layer has a plurality ofmicrofluidic reaction openings distributed throughout the layer. Anoligonucleotide probe is preferably bound to the polymer layer.

[0014] U.S. Pat. No. 6,127,127 provides a self-assembled phosphonatemonolayer, which in the preferred embodiment is a carboxy-alkylphosphonate, on an ITO surface. The oligonucleotide probe is immobilizedon an electrode surface modified by the self-assembled monolayer. Theelectrode with the self-assembled monolayer is useful for theelectrochemical detection of a preselected base in a nucleic acid andfor determining the presence of a target nucleic acid in a sample, bycontacting the self-assembled monolayer with the sample, so that thetarget nucleic acid and the oligonucleotide probe form a hybridizednucleic acid on the monolayer; reacting the hybridized nucleic acid witha transition metal complex capable of oxidizing a preselected base inthe hybridized nucleic acid in an oxidation-reduction reaction;detecting the oxidation-reduction reaction; and determining the presenceor absence of the target nucleic acid from the detectedoxidation-reduction reaction.

[0015] In both the polymer-electrode and monolayer patents,determination of the presence of a target protein in a sample can alsobe achieved and comprises attaching a protein-binding substance to apolymer-electrode or self-assembled monolayer on a conductive workingsurface according to the invention; exposing the polymer-electrode ormonolayer to the sample; exposing the polymer-electrode or monolayer toa second protein-binding substance that has been modified to contain anoligonucleotide label; reacting the polymer-electrode or monolayer witha transition metal complex capable of oxidizing a preselected base inthe oligonucleotide label in an oxidation-reduction reaction; detectingthe oxidation-reduction reaction; and determining the presence orabsence of the target protein from the detected oxidation-reductionreaction. The. polymer-electrode or monolayer may be brought intocontact with the conductive working surface of the substrate eitherbefore or after reacting the polmer-electrode or monolayer with thefirst protein binding substance. The target protein may be modified tocontain an oligonucleotide label as is known in the art.

[0016] Amino acids, such as tyrosine and tryptophan, have been detectedby direct, unmediated electrochemistry (Brabec, V. and Mornstein, V.,Biochimica et Biophysica Acta, 1980, 625, 43; Renaud, J. A. et al.,Bioelectrochem, & Bioenergetics, 1980, 7, 395). However, the levels ofcurrent obtained by direct, unmediated oxidation of amino acids aregenerally low, on the order of a few microamps for concentratedsolutions of amino acids (100 μM).

[0017] Oxidation potentials of several amino acids have been determinedusing thermodynamic and kinetic methods (DeFilippis, M. R et al.,Biochem., 1989, 28, 4857). The oxidation potential for tyrosine is about0.6-0.73 V (vs. Ag/AgCl reference), and the oxidation potential fortryptophan is about 0.6-0.85 V (vs. Ag/AgCl). Other amino acid oxidationpotentials have been estimated for histidine (1.1-1.4 V), cysteine(0.5-0.8 V), methionine (0.9-1.2 V), and cystine (1.1-1.2 V)(Brabec, V.and Mornstein, V., Biochimica et Biophysica Acta, 1980, 625, 43; Renaud,J. A. et al., Bioelectrochem. & Bioenergetics, 1980, 7, 395), but theextent to which these amino acids are oxidized depends on the electrodematerial.

[0018] It has been observed that proteins can be adsorbed to electrodesat both negative and positive potentials through an electrostaticinteraction when the protein net charge is opposite that of theelectrode (Brabec, V. et al., Bioelectrochem. & Bioenergetics, 1981, 8,451). Although the adsorption is initiated by an electrostaticinteraction, it has been found that adsorption in this manner leads tothe formation of a protein that is irreversibly adsorbed to theelectrode surface. This phenomenon can interfere with electrochemicaldetection at electrodes because the adsorbed protein blocks theelectrode surface. Thus, in attempts to detect protein in solutiondirectly, Brabec et al. found that the adsorbed protein fouled theelectrode surface so that fresh protein molecules could not teach thesurface and interfered with protein detection at the electrode. Alongthese same lines, Elbicki et al. (Elbicki, J. et al., Biosensors, 1989,4, 251) have found that the removal of proteins from samples isnecessary to protect electrode surfaces from fouling.

[0019] Because of the low sensitivity and poor selectivity of directelectrochemical detection of proteins, attempts have been made to useexogenous labels to facilitate this electrochemical detection. In oneexample, DiGleria et al. sought to convert a “redox-inactive” protein toa “redox-active’ protein by adding an exogenous redox-active label tothe enzyme β-lactamase (DiGleria, K et al., FEBS Letters, 1997, 400,155). This approach involved engineering the enzyme to contain anunnatural cysteine residue and then modifying this residue with athiol-reactive ferrocene compound, N-(2-ferrocene-ethyl)maleimide.

[0020] Previous work With metal complexes and amino acids includes thestudy of one-electron oxidation of tryptophan by ruthenium-DNAintercalator compounds (Wagenknecht, H.-A. et al., J. Amer. Chem. Soc.,2000, 122, 1). This process is not catalytic, however, and was initiatedby light rather than by an applied electrochemical potential. Also, inthis system, electron transfer between tryptophan and the rutheniumcomplex was found to be dependent on guanine as an intermediate. Inanother study, the one-electron oxidation of a reference redox couple,osmium²⁺ (2,2′-bipyridine)³ (Os(bpy)₃), was used to determine the redoxpotentials of tryptophan and tyrosine by pulse radiolysis (DeFilippis,M. R. et al. Biochem., 1989, 28, 4857), but in this case, Os(bpy)₃ ²⁺acted as a reductant of an oxidized amino acid. Therefore, a two-stepcatalytic oxidation reaction between oxidized transition metal complexand the amino acid was not present in this prior work.

[0021] Recently, Pikulski and Gorski have suggested the catalyticoxidation of disulfide bonds and the amino acid cystine by the iridiumcomplex, Ir(H₂O)₂Cl₂ (Pikulski, M. et al., Anal. Chem., 2000, 72, 2696).This chemistry has been utilized to create a flow injection sensor fordetecting insulin, in which the mediator is immobilized in an oxidelayer on a glassy carbon electrode and the insulin is in solution. Arelated method has been proposed for detecting amino acids and peptidesin solution at copper electrodes using catalytic electrochemistry(Brazill, S. A. et al., Anal. Chem., 2000, 72, 5542). Although not fullyunderstood, the detection process is believed to involve amino acidoxidation by Cu(III) in a conductive oxide or hydroxide layer that isformed under alkaline conditions. In both the work of Pikulski et al.and Brazill et al., the metal complex is a solid phase mediator in theelectrode to which the analyte diffuses nonspecifically. Thus, theelectrode is only capable of detecting analytes that come into directcontact with the electrode. The electrochemical detection utilized inthe above methods is distinct from the mediated electrochemicaldetection methods of the instant invention wherein there is anonconductive layer and wherein the mediator is a soluble, freelydiffusible transition metal complex that is capable of oxidizing labelson binders that are bound to the electrode via biological bindinginteractions (antibody-protein, receptor-ligand, DNA-protein, andprotein-protein).

[0022] A means of detecting protein binding has been disclosed in thePCT application of Fowlkes and Thorp using a soluble transition metalcomplex mediator and biomolecules labeled with transition metalcomplexes including Ru(bpy)₃ ²⁺ and OS(bpy)₃ ²⁺ (PCT/US98/02440). Thebasic mechanism of this detection scheme involves electron transfer fromthe label to an electrode via the soluble mediator. The electrochemicalcurrent enhancement obtained from the label is limited by the number ofelectrons in the label that can be oxidized by the soluble mediator sothe oxidation of the mediator is limited to only one cycle per label forRu(bpy)₃ ²⁺ and Os(bpy)₃ ²⁺. The method of Fowlkes and Thorp isdistinctly different from the technology described in this applicationin that the electrochemical label is a transition metal complex inFowlkes and Thorp, whereas the present invention provides a differentlabel. When the electrochemical label is a transition metal complex, thenumber of cycles of mediated electron transfer is generally limited toone per label.

[0023] Measurement of a target protein was first achieved by Yalow andBerson (Yalow, R. S. and Berson, S. A., Nature, 1959, 184, 1648) using acompetitive, radiolabeled ligand immunoassay (i.e., RIA) for the proteininsulin. Since then numerous other labels have been employed inimmunoassays such as enzymes, chemiluminescent and fluorescent labels,metal atoms, transition metal complexes and particles (i.e.,polystyrene, gold) to measure selected target proteins (Tijssen, P., In:Laboratory Techniques in Biochemistry and Molecular Biology, 1985, Vol.15. Elsevier Science Publishers, N.Y. 549 p). More recently, numerousother types of macromolecules other than immunoglobulins such as cellreceptors (Guyda, H. J., J. Clin. Endocrinol. Metab., 1975, 41, 953;Strosberg, A. D., et al., Curr. Opin. Biotechnol., 1991, 2, 30),proteoglycans (Najam, S., Cytokine, 1997, 9, 1013), extracellular matrixproteins (Mould, A. P., Meth. Mol. Biol., 2000, 139, 295) and nucleicacids (McGown L. B., et al., Anal. Chem., 1995, 67, 663A) have been usedas affinity binders in assays to detect target proteins. The instantinvention to be described herein has the ability to utilize all theabove molecular interactions to detect preselected target proteins aswell as other binding substances by using peptide/amino acid oroligonucleotide labels in assays with catalytic transition metalmediated electrochemical detection.

[0024] Recently developed protein detection assay systems arecommercially available from IGEN (Gaithersburg, Md.) and Luminex(Austin, Tex.). However, each of these systems is readilydistinguishable from the present invention since they are dependent uponthe teachings of electrochemiluminescence (IGEN) or fluorescent beadsorting (Luminex).

[0025] It is an object of this invention to detect binding interactions.The detection methodology involves oxidation of labels using transitionmetal mediator complexes in a detectable catalytic redox reaction andapplies generally to binding interactions of immunoglobulins, receptors,proteins, and oligonucleotides with proteins, protein fragments,ligands, carbohydrates, drugs, drug candidates, steroids, hormones, andother substances. The labels are attached directly to the binders,target molecules, surrogate target molecules or to binders capable ofbinding targets or surrogate target molecules that compete with thetarget for binding. The labels can be naturally attached in the target,surrogate target, or binder (i.e., endogenous) or constructed by thecovalent attachment of the label to the target, binder, or surrogatetarget (i.e., exogenous). Preferred labels include peptides andoligonucleotides. Preferred types of binding affinities to be utilizedin the instant invention to detect target substances are based onantibody—antigen, receptor (eukaryotic, prokaryotic or viral)—ligand,DNA—protein, drug target—drug, and protein—protein interactions.

[0026] It is a further object of this invention to use detected bindinginteractions between specific biomolecules to determine their presence(or absence) in test samples, such as clinical samples, environmentalsamples, pharmaceutical samples and others. It is an additional objectof this invention to determine the impact of specific drugs on thedetected binding interactions between biomolecules.

[0027] Other objects and advantages will be more fully apparent from thefollowing. disclosure and appended claims.

SUMMARY OF THE INVENTION

[0028] The invention herein provides a method of detecting bindinginteractions and target molecules, such as proteins, protein fragments,recombinant proteins, recombinant protein fragments, extracellularmatrix proteins, ligands, carbohydrates, steroids, hormones, drugs, drugcandidates, immunoglobulins and receptors of eukaryotic, prokaryotic orviral origin, by mediated electrochemistry using labels that react withtransition metal mediator complexes in a detectable catalytic redoxreaction. These labels are attached directly to binders, targetmolecules, surrogate target molecules, or to affinity ligands capable ofbinding to the target or to surrogate target molecules capable ofcompeting with the target for binding to another binder. The labels canbe naturally present (endogenous) in the binder, target or affinityligand, or constructed by the covalent attachment of the label to thebinder, target, affinity ligand or surrogate target (exogenous). Thebiological binding interactions detected with this technology includeantibody-antigen, protein-protein, protein-nucleic acid, drugtarget-drug, and receptor-ligand interactions.

[0029] Other objects and features of the inventions will be more fullyapparent from the following disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIGS. 1A-1C. Electrochemical detection of amino acids in solutionusing mediated catalytic electrochemistry. Cyclic voltammograms (300mV/s) were collected of three different transition metal mediators (FIG.1A: Ru(bpy)₃ ²⁺, FIG. 1B: Os(bpy)₃ ²⁺ and FIG. 1C: Os(Me₂-bpy)₃ ²⁺) at aconcentration of 20 μM in 50 mM phosphate buffer, pH 7.5 in the presenceof 100 μM tryptophan (

), 100 82 M tyrosine ( . . . ), 100μM 5-hydroxytryptophan (

), or no amino acid (- - -)

[0031]FIGS. 2A-2B. Electrochemical detection of passively adsorbed mouseIgG (FIG. 2A), but not human IgG (FIG. 2B), by the increased currentgenerated following the sequential binding of two binders (i.e.,antibodies). Following passive adsorption of either mouse or human IgGfor 15 hr, ITO electrodes were blocked with 0.1% polyvinyl alcohol andwere treated with a goat anti-mouse IgG (15 μg/ml) that had beenaffinity isolated on a mouse IgG column and subsequently adsorbed withhuman IgG to remove any cross reactivity to human IgG. This antibody wasremoved after a 2 hr. S incubation, the ITO washed with PBS and thenexposed to the second binder (rabbit anti-goat IgG at 15 μg/ml) for anadditional 2 hr. Increased current generated over that seen by theoxidation of the passively adsorbed mouse IgG alone and over that seenwith adsorbed human IgG after sequential incubation with both solublebinders was indicative of additional amino acid oxidation due to bindingof both soluble binders when electrodes were coated with mouse IgG.Current changes were assessed using cyclic voltammetry (2.5 V/s) and theredox mediator, Ru(bpy)₃ ²⁺, at 50 μM in 50 mM sodium phosphate, pH 7.0.Numbering of Treatment Sequences in FIG. 2. FIG. 2A: #1, PVA block only;#2, mouse IgG→PVA block; #3, mouse IgG→PVA block→first binder; #4, mouseIgG→PVA block→first binder→second binder. FIG. 2B: #1, PVA block only;#2, human IgG→PVA block; #3, human IgG→PVA block→first binder; #4, humanIgG→PVA block→first binder→second binder.

[0032]FIG. 3. Detection of human chorionic gonadotropin (hCG) hormoneusing an oligonucleotide-labeled antibody and mediated catalyticelectrochemistry. ITO electrodes were modified by passively adsorbing 20μg/ml rabbit anti-beta chain hCG IgG in 100 mM acetate buffer, pH 5.0,overnight, and were blocked with 0.1% polyvinyl alcohol. The modifiedelectrodes were treated with 0, 10, or 50 ng/ml hCG, washed withphosphate buffered saline, and treated with 25 μg/ml goat anti-alphachain hCG IgG labeled with five guanine-containing oligonucleotidelabels per IgG in phosphate buffered saline. After washing theelectrodes, cyclic voltammograms (2.5 V/s) were collected in thepresence of 50 μM Ru(bpy)₃ ²⁺ in 50 mM phosphate buffer, pH 7.0.Numbering of Treatment Sequences in FIG. 3. #1, PVA block only, #2,rabbit anti-beta chain hCG IgG→PVA block→no hCG→goat anti-alpha chainhCG IgG labeled with oligonucleotide; #3, rabbit anti-beta chain hCGIgG→PVA block→50 ng/ml hCG→goat anti-alpha chain hCG IgG; #4, rabbitanti-beta chain hCG IgG→PVA block→10 ng/ml hCG→goat anti-alpha chain hCGIgG labeled with oligonucleotide; #5, rabbit anti-beta chain hCG IgG→PVAblock 50 ng/ml hCG goat anti-alpha chain hCG IgG labeled witholigonucleotide.

[0033]FIG. 4A-4B. Detection of human chorionic gonadotropin (hCG)hormone using a peptide-labeled antibody and mediated catalyticelectrochemistry. ITO electrodes were modified by passively adsorbing 20μg/ml rabbit anti-beta chain hCG IgG in 100 mM acetate buffer, pH 5.0,overnight, and were blocked with 0.1% polyvinyl alcohol. The modifiedelectrodes were treated with 100 ng/ml hCG, washed with phosphatebuffered saline, and treated with 30 μg/ml goat anti-alpha chain hCG IgGlabeled with five tyrosine-containing peptide labels per IgG inphosphate buffered saline for 1.5 hr. After washing the electrodes,cyclic voltammograms (2.5 V/s) were collected in the presence of 50 μMOs(bpy)₃ ²⁺ in 50 mM phosphate buffer, pH 7.0. Numbering of TreatmentSequences in FIG. 4. FIG. 4A: #1, PVA block only, #2, rabbit anti-betachain hCG IgG→PVA block; #3, rabbit anti-beta chain hCG IgG→PVA block→nohCG→goat anti-alpha chain hCG IgG labeled with peptide; #4, rabbitanti-beta chain hCG IgG→PVA block 100 ng/ml hCG goat anti-alpha chainhCG IgG labeled with peptide. FIG. 4B: #1, PVA block only; #2, rabbitanti-beta chain hCG IgG→PVA block; #3, rabbit anti-beta chain hCGIgG→PVA block→no hCG→goat anti-alpha chain hCG IgG; #4, rabbit anti-betachain hCG IgG→PVA block→100 ng/ml hCG→goat anti-alpha chain hCG IgG.

[0034]FIG. 5A-5C. Detection of an affinity captured IgG labeled with apeptide containing the low potential amino acid 5-hydroxytryptophan. ITOelectrodes were modified by passively adsorbing 20 μg/ml rabbitanti-goat IgG (Sigma Chemical) (FIG. 5A,C) or, as a control, 20 μg/mlnon-immune rabbit IgG (Sigma Chemical) (FIG. 5B) in 50 mM acetatebuffer, pH 5, for 15 hr. All the ITO electrodes were blocked with 0.1%polyvinyl alcohol. After washing, electrodes were exposed to 25 μg/mlpeptide-labeled goat IgG (IgG-WOH) (FIG. 5A,B) or the same unlabeled IgG(FIG. 5C). The peptide used for labeling was prepared by The AmericanPeptide Co., Inc., Sunnyvale, Calif. Cyclic voltammograms (2.5 V/sec)were collected in duplicate in the presence of 50 μm Os(Me₂-bpy)₃ ²⁺ in50 mM phosphate buffer. Numbering of Treatment Sequences in FIG. 5. FIG.5A: #1, PVA block only; #1, rabbit anti-goat IgG→PVA block; #3, rabbitanti-goat IgG→PVA block goat IgG-WOH; FIG. 5B: #1, PVA block only;#2rabbit IgG (non-immune)→PVA block; #3, rabbit IgG (non-immune)→PVAblock→goat IgG-WOH; FIG. 5C: #1, PVA block only, #2, rabbit anti-goatIgG→PVA block; #3, rabbit anti-goat IgG→PVA block→goat IgG.

[0035]FIG. 6. Competitive assay for detection of biotin (vitamin H) in asample using biotin labeled with a peptide containing5-hydroxytryptophan. ITO electrodes were modified by passively adsorbing20 μg/ml neutravidin in 100 mM acetate buffer, pH 5.0 overnight and wereblocked with 0.1% polyvinyl alcohol. The modified electrodes weretreated with 1 μM biotin-peptide (

) in phosphate buffered saline, 1 μM biotin-peptide plus 160 μM freebiotin (- - -) in phosphate buffered saline, or phosphate bufferedsaline alone ( . . . ). After washing the electrodes, cyclicvoltammograms (2.5 V/s) were collected in the presence of 50 μMOs(Me₂-bpy)₃ ²⁺ in 50 mM phosphate buffer, pH 7.3. The 21-mer peptidelabel contained three 5-hydroxytryptophan residues.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

[0036] The present invention provides a method of detecting bindinginteractions by mediated electrochemistry using labels that react withtransition metal mediator complexes in a detectable catalytic redoxreaction. These labels are attached directly to binders, targetmolecules, surrogate targets, or affinity binders capable of binding tothe target or to surrogate targets. The labels can be naturally present(endogenous) in the binder, target or affinity ligand, or constructed bythe covalent attachment of the label to the binder, target, affinityligand or surrogate target (exogenous). In the present invention suchexogenous labels may be oligonucleotides or peptides containing aminoacids capable of being oxidized in an oxidation-reduction reaction. Mostpreferably, such a peptide label contains one or more amino acidscapable of being oxidized in an oxidation-reduction reaction atapproximately ≦0.6V, and the transition metal mediator isosmium²⁺(4,4′-dimethyl-2,2′-bipyridine)₃.

[0037] The biological binding interactions detected with this technologyinclude but are not limited to antibody-antigen, protein-protein,protein-nucleic acid, drug target-drug, and receptor-ligandinteractions. The binding interactions can be detected in severalformats including sandwich, competitive, and target-bound assays. Thetarget substance can generally be any substance that one might wish todetect, generally being selected from the group consisting of proteins,protein fragments, ligands, carbohydrates, drugs, drug candidates andhormones. The specific target substance for a particular assay willdepend on the nature of that assay. Thus, for an assay geared to smallermolecules, such as for drugs or drug candidates, a competitive assaymight be preferred, while for an assay geared to larger molecules, asandwich assay might be preferred.

[0038] As discussed in more detail below, the invention utilizes anelectrode comprising a conductive substrate modified with anon-conductive layer, through which non-conductive layer a transitionmetal complex can transfer electrons to the conductive substrate. Theelectrode further comprises a member of a biological binding pairimmobilized to the electrode, which member of the biological bindingpair is capable of specifically binding another specific biologicalbinder. The nonconductive layer may be the immobilized binder or aseparate non-conductive layer on which the binder may be immobilized asis known in the art. For some uses, the nonconductive layer may beselected from the group consisting of streptavidin, avidin, protein A,protein G, and antibodies. It may also be a silane molecule covalentlyattached to the conductive substrate, said silane molecule further beingcapable of forming a covalent bond with the binder. The nonconductivelayer to which the binder is immobilized may comprise one or morecomponents.

[0039] Following is a summary of the various preferred embodiments ofthe invention, after which is a detailed discussion of components of theinvention. In particular, the invention herein includes a firstembodiment for a method of determining the presence or absence of atarget substance in a test sample, comprising:

[0040] a) providing an electrode comprising a conductive substratemodified with a non-conductive layer having an immobilized first bindercapable of binding the target substance and through which layer atransition metal mediator can freely move to transfer electrons to theconductive substrate;

[0041] b) contacting the immobilized first binder with the test sampleto form a target complex if the target substance is present in the testsample;

[0042] c) contacting the first binder or the target complex, if present,with a second binder capable of binding the target substance and havingan endogenous or exogenous label capable of being oxidized in anoxidation-reduction reaction;

[0043] d) contacting the electrode, the immobilized first binder, andthe target complex having the second binder, if present, with atransition metal mediator that oxidizes the label in anoxidation-reduction reaction between the transition metal mediator andthe label, from which label there is electron transfer to the transitionmetal mediator resulting in regeneration of the reduced form of thetransition metal mediator as part of a catalytic cycle;

[0044] e) detecting the oxidation-reduction reaction; and

[0045] f) determining the presence or absence of the target substance inthe test sample from the detected oxidation-reduction reaction.

[0046] The target substance in this embodiment of the invention is mostpreferably a protein. The immobilized first binder is preferablyselected from the group consisting of immunoglobulins, receptors,proteins, such as an extracellular matrix protein, and oligonucleotides.The immobilized first binder may be either a synthetic or natural (e.g.,of eukaryotic; prokaryotic or viral origin) molecule. The test sampleand the second binder may be added to the immobilized first bindersimultaneously.

[0047] In another embodiment of the invention herein, the invention is amethod of determining the presence or absence of a target substance in atest sample, comprising:

[0048] a) providing an electrode comprising a conductive substratemodified with a non-conductive layer having an immobilized bindercapable of binding the target substance and through which layer atransition metal mediator can freely move to transfer electrons to theconductive substrate;

[0049] b) contacting the immobilized binder with the test sample to forma target complex if the target substance is present in the test sample;

[0050] c) contacting the immobilized binder with an endogenously orexogenously labeled substance capable of binding with the immobilizedbinder, such that binding of the labeled substance is inhibited if thetarget complex is present, and wherein the label is capable of beingoxidized in an oxidation-reduction reaction;

[0051] d) contacting the electrode, the immobilized binder, the targetsubstance, and the labeled substance, if present, with a transitionmetal mediator that oxidizes the label in an oxidation-reductionreaction between the transition metal mediator and the label, from whichlabel there is electron transfer to the transition metal mediatorresulting in regeneration of the reduced form of the transition metalmediator as part of a catalytic cycle;

[0052] e) detecting the oxidation-reduction reaction; and

[0053] f) determining the presence or absence of the target substance inthe test sample from the detected oxidation-reduction reaction.

[0054] This embodiment is a competitive assay where the test sample andlabeled substance may be added to the immobilized binder eithersequentially or simultaneously. The labeled substance in this embodimentis preferably selected from the group consisting of proteins, proteinfragments, recombinant proteins and recombinant protein fragments,ligands, carbohydrates, drugs, drug candidates, steroids and hormones.

[0055] In a third embodiment of the invention, the invention is a methodof determining the presence or absence of a target substance in a testsample, comprising:

[0056] a) providing an electrode comprising a conductive substratemodified with a non-conductive layer having an immobilized bindercapable of binding the target substance and through which layer atransition metal mediator can freely move to transfer electrons to theconductive substrate;

[0057] b) contacting the immobilized binder with a surrogate targetcapable of binding with the immobilized binder to form a target complex,said surrogate target having an endogenous or exogenous label capable ofbeing oxidized in an oxidation-reduction reaction;

[0058] c) contacting the target complex with the test sample, so thatlabeled surrogate target is displaced from the immobilized binder by thetarget substance, if the target substance is present in the test sample;

[0059] d) contacting the electrode, the immobilized binder, and saidsurrogate target, if present, with a transition metal mediator thatoxidizes the label in an oxidation-reduction reaction between thetransition metal mediator and the label, from which label there iselectron transfer to the transition metal mediator resulting inregeneration of the reduced form of the transition metal mediator aspart of a catalytic cycle;

[0060] e) detecting the oxidation-reduction reaction; and

[0061] f) determining the presence or absence of the target substance inthe test sample from the detected oxidation-reduction reaction.

[0062] This embodiment of the invention utilizes a surrogate targethaving an endogenous or exogenous label, and the surrogate target ispreferably selected from the group consisting of proteins, proteinfragments, recombinant proteins and recombinant protein fragments,ligands, carbohydrates, drugs, drug candidates, steroids and hormones.

[0063] In a fourth embodiment of the invention herein, the invention isa method of determining the presence or absence of a target substance ina test sample comprising:

[0064] a) providing an electrode comprising a conductive substratemodified with a non-conductive layer having an immobilized targetsubstance or an immobilized surrogate target substance, and throughwhich layer a transition metal mediator can freely move to transferelectrons to the conductive substrate;

[0065] b) contacting the immobilized target substance or immobilizedsurrogate target substance with the test sample and with an endogenouslyor exogenously labeled binder that will bind the target substance in thetest sample such that the target substance in the test sample, ifpresent, competes with the immobilized target substance or theimmobilized surrogate target substance for the labeled binder, saidlabel being capable of being oxidized in an oxidation-reductionreaction;

[0066] c) contacting the electrode, the immobilized target substance orimmobilized surrogate target substance, and the labeled binder, ifpresent, with a transition metal mediator that oxidizes the label in anoxidation-reduction reaction between the transition metal mediator andthe label, from which label there is electron transfer to the transitionmetal mediator resulting in regeneration of the reduced form of thetransition metal mediator as part of a catalytic cycle;

[0067] d) detecting the oxidation-reduction reaction; and

[0068] e) determining the presence or absence of target substance in thetest sample from the detected oxidation-reduction reaction.

[0069] The labeled binder and the test sample may be mixed prior tobeing added to the immobilized target substance or immobilized surrogatetarget substance. The nonconductive layer may be the immobilized targetsubstance or the immobilized surrogate target substance, or may compriseother molecules.

[0070] In a fifth embodiment of the invention herein, the invention is amethod of determining the effect of a test sample on the bindinginteractions between two binders that are members of a binding pair,said method comprising:

[0071] a) providing an electrode comprising a conductive substratemodified with a non-conductive layer having an immobilized first binderand through which layer a transition metal mediator can freely move totransfer electrons to the conductive substrate;

[0072] b) contacting the immobilized first binder with the test sample;

[0073] c) contacting the immobilized first binder with an endogenouslyor exogenously labeled second binder for said first binder, said labelbeing capable of being oxidized in an oxidation-reduction reaction;

[0074] d) contacting the electrode, the immobilized first binder, andthe labeled second binder, if present, with a transition metal mediatorthat oxidizes the label in an oxidation-reduction reaction between thetransition metal mediator and the label, from which label there iselectron transfer to the transition metal mediator resulting inregeneration of the reduced form of the transition metal mediator aspart of a catalytic cycle;

[0075] e) detecting the oxidation-reduction reaction; and

[0076] f) determining the effect of the test sample on the ability ofthe second binder to bind to the first binder from the detectedoxidation-reduction reaction.

[0077] In this embodiment, the test sample, the first binder and thesecond binder may each be selected from the group consisting ofproteins, protein fragments, recombinant proteins, recombinant proteinfragments, extracellular matrix proteins, ligands, carbohydrates,steroids, hormones, drugs, drug candidates, immunoglobulins, receptorsof eukaryotic, prokaryotic or viral origin, and oligonucleotides. Thetest sample and labeled second binder may be added to the immobilizedfirst binder simultaneously or the labeled second binder may be added tothe immobilized first binder before the addition of the test sample todetermine the effect of the test sample on the binding interactionsbetween the first binder and the second binder.

[0078] In a sixth embodiment of the method of the invention herein, theinvention is a method of determining the presence or absence of a targetprotein in a test sample, said target protein having an endogenous labelcapable of being oxidized in an oxidation-reduction reaction,comprising:

[0079] a) providing an electrode comprising a conductive substratemodified with a non-conductive layer having an immobilized bindercapable of binding the target protein and through which layer atransition metal mediator can freely move to transfer electrons to theconductive substrate;

[0080] b) contacting the immobilized binder with the test sample to forma target complex if the target protein is present in the test sample;

[0081] c) contacting the electrode, the immobilized binder and thetarget protein, if present, with a transition metal mediator thatoxidizes the label in an oxidation-reduction reaction between thetransition metal mediator and the label, from which label there iselectron transfer to the transition metal mediator resulting inregeneration of the reduced form of the transition metal mediator aspart of a catalytic cycle;

[0082] d) detecting the oxidation-reduction reaction; and

[0083] e) determining the presence or absence of the target protein inthe test sample from the detected oxidation-reduction reaction.

[0084] In this embodiment, the target is a protein having an endogenouslabel.

[0085] The invention herein further comprises a labeled member of abinding pair useful for mediated catalytic electrochemistry, whichcomprises:

[0086] a) a binder selected from the group consisting of proteins,protein fragments, recombinant proteins, recombinant protein fragments,extracellular matrix proteins, ligands, carbohydrates, steroids,hormones, drugs, drug candidates, immunoglobulins, receptors ofeukaryotic, prokaryotic or viral origin, and oligonucleotides; and

[0087] b) an exogenous peptide label containing one or more modifiedamino acids capable of being oxidized in an oxidation-reduction reactionat potentials below those of naturally occurring amino acids.

[0088] The preferred binder in this member of a binding pair is anantibody. There are at least two modified amino acids in the peptidelabel. In the preferred embodiment, the modified amino acids in thepeptide label are selected from derivatives of tyrosine and tryptophan,such as 5-hydroxytryptophan; 3-aminotyrosine; and3,4-dihydroxyphenylalanine.

[0089] A. Mediators and Oxidation-Reduction Reactions

[0090] The mediator that is needed to enable electron transfer may beany molecule such as a cationic, anionic, non-ionic, or zwitterionicmolecule that is reactive with the electrochemical label at a uniqueoxidation potential to transfer electrons from the label to theelectrode. It is important that the mediators used in the inventionherein be selected to exhibit a reversible redox couple at about thesame oxidation potential or higher than that observed for the label thatis being detected. Thus, to use tyrosine or tryptophan as the label, themediator must have an oxidation potential of about ≧0.65 V or ≧0.8 V vs.Ag/AgCl, respectively. Suitable mediators would be Os(bpy)₃ ²⁺ andFe(bpy)₃ ²⁺, respectively. Similarly, in order to use guanine as thelabel, the mediator must have an oxidation potential about ≧1.1 V vs.Ag/AgCl, and an appropriate mediator is Ru(bpy)₃ ²⁺. Other examples ofsuitable mediators for use in the methods of the present invention aretransition metal complexes, including, for example,Ruthenium²⁺(2,2′-bipyridine)₃ (“Ru(bpy)₃ ²⁺”);Ruthenium²⁺(4,4′-dimethyl-2,2′-bipyridine)₃ (“Ru(Me₂-bpy)₃ ²⁺”);Ruthenium²⁺(5,6-dimethyl-1,10-phenanthroline)₃ (“Ru(Me₂-phen)₃ ²⁺”);Iron²⁺(2,2′-bipyridine)₃ (“Fe(bpy)₃ ²⁺”);Iron²⁺(4,4′-dimethyl-2,2′-bipyridine)₃ (“Fe(Me₂-bpy)₃ ²⁺”);Iron²⁺(5-chlorophenanthroline)₃ (“Fe(5-Cl-phen)₃ ²⁺”);Iron²⁺(4,4′-dimethyl-2,2′-bipyridine)(bipyridine)₂ (“Fe(Me₂-bpy)(bpy)₂²⁺”);Iron²⁺(4,4′-dimethyl-2,2′-bipyridine)₂(bipyridine)(“Fe(Me₂-bpy)₂(bpy)²⁺”);Osmium²⁺(2,2′-bipyridine)₃ (“Os(bpy)₃ ²⁺”);Osmium²⁺(4,4′-dimethyl-2,2′-bipyridine)₃ (“Os(Me₂-bpy)₃ ²⁺”;Osmium²⁺(5-chlorophenanthroline)₃ (“Os(5-Cl-phen)₃ ²⁺”);Osmium²⁺(4,4′-dimethyl-2,2′-bipyridine)(bipyridine)₂ (“Os(Me₂-bpy)(bpy)₂²⁺”); Osmium²⁺(4,4′dimethyl-2,2′-bipyridine)₂(bipyridine)(“Os(Me₂-bpy)₂(bpy)²⁺”); dioxorhenium¹⁺phosphine; anddioxorhenium¹⁺pyridine (“ReO₂(py)₄ ¹⁺”). Some anionic complexes usefulas mediators are: Ru(bpy)((SO₃)₂-bpy)₂ ²⁻ and Ru(bpy)((CO₂)₂-bpy)₂ ²⁻and some zwitterionic complexes useful as mediators areRu(bpy)₂((SO₃)₂-bpy) and Ru(bpy)₂((CO₂)₂-bpy) where (SO₃)₂-bpy²⁻ is4,4′-disulfonato-2,2′-bipyridine and (CO₂)₂-bpy²⁻ is4,4′-dicarboxy-2,2′-bipyridine. Suitable substituted derivatives of thepyridine, bipyridine and phenanthroline groups may also be employed incomplexes with any of the foregoing metals. Suitable substitutedderivatives include but are not limited to 4-aminopyridine;4-dimethylpyridine; 4-acetylpyridine; 4-nitropyridine;4,4′-diamino-2,2′-bipyridine; 5,5′-diamino-2,2′-bipyridine;6,6′-diamino-2,2′-bipyridine; 5,5′-dimethyl-2,2′-bipyridine;6,6-dimethyl-2,2′-bipyridine; 4,4′-diethylenediamine-2,2′-bipyridine;5,5′-diethylenediamine-2,2′-bipyridine;6,6′-diethylenediamine-2,2′-bipyridine; 4,4′-dihydroxyl-2,2′-bipyridine;5,5′-dihydroxyl-2,2′-bipyridine; 6,6′-dihydroxyl-2,2′-bipyridine;4,4′,4″-triamino-2,2′,2″-terpyridine;4,4′,4″-triethylenediamine-2,2′,2″-terpyridine;4,4′,4″-trihydroxy-2,2′,2″-terpyridine;4,4′,4″-trinitro-2,2′,2″terpyridine;4,4′,4″-triphenyl-2,2′,2″-terpyridine; 4,7-diamino-1,10-phenanthroline;3,8-diamino-1,10-phenanthroline;4,7-diethylenediamine-1,10-phenanthroline;3,8-diethylenediamine-1,10-phenanthroline;4,7-dihydroxyl-1,10-phenanthroline; 3,8-dihydroxyl-1,10-phenanthroline;4,7-dinitro-1,10-phenanthroline; 3,8-dinitro-1,10-phenanthroline;4,7-diphenyl-1,10-phenanthroline; 3,8-diphenyl-1,10-phenanthroline;4,7-disperamine-1,10-phenanthroline;3,8-disperamine-1,10-phenanthroline; dipyrido[3,2-a:2′,2′-c]phenazine;4,4′-dichloro-2,2-bipyridine; 5,5′-dichloro-2,2′-bipyridine; and6,6′-dichloro-2,2′-bipyridine.

[0091] B. Oxidation-Reduction Reaction.

[0092] The mediator may be reacted with labels in or on the capturedtarget, the surrogate target, or the binder under conditions sufficientto effect the oxidation-reduction reaction of the mediator with thelabel via a catalytic reaction. The solution in which theoxidation-reduction reaction takes place may be any suitable solutionfor solubilizing the components of the assay and preferably compriseswater. Suitable conditions for permitting the oxidation-reductionreaction to occur will be known to those skilled in the art.

[0093] C. Detection of Oxidation-Reduction Reactions The occurrence ofthe oxidation-reduction reaction of the invention may be detectedaccording to any suitable means known to those skilled in the art. Forexample, the occurrence of the oxidation-reduction reaction may bedetected using a detection (working) electrode to observe a change inthe electrochemical signal, which is indicative of the occurrence of theoxidation-reduction reaction. An electrode suitable for the detection oflabels in accordance with the methods described herein comprises aconductive substrate having a working surface thereon, and is sensitiveto the transfer of electrons between the mediator and the label Theconductive substrate may be a metallic substrate or a non-metallicsubstrate, including semiconductor substrates. Preferably the electrodeis a tin-doped indium oxide (ITO) electrode, a tin-oxide or an indiumoxide electrode. Alternatively, the electrode may be of gold, carbonfiber or glassy carbon. The suitability of a particular electrodematerial ultimately is dependent on the utility of that material withthe selected label(s) and mediator(s) at their required redoxpotentials. The conductive substrate may take any physical form, such asan elongate probe having a working surface formed on one end thereof, ora flat sheet having the working surface on one side thereof, for examplein the wells of a microtiter plate.

[0094] In order to prepare the electrode for modification withimmobilized biological binding entities, the electrode is modified witha suitable nonconductive layer. The nonconductive layer may have one ormore of a number of functions including providing covalent attachment ofbiomolecules, blocking of nonspecific binding to the electrode, andallowing electron transfer between the mediator and the electrode and/orthe mediator and the label. The nonconductive layer may be one or moreof the following, for example: self-assembled monolayers (e.g., U.S.Pat. No. 6,127,127); cross-linked polymer layers; alkyl silane layers;alkylphosphonate-, alkylphosphate-, carboxyalkane-, alkanethiol-, oralkylamine-based layers; polymer membranes (as in U.S. Pat. No.5,968,745) and/or one or more layers of biomolecules such as proteins,antibodies, biotin-binding molecules (avidin, streptavidin,neutravidin), protein A, protein G, receptors, or oligonucleotides. Inthe case of a nonconductive layer comprised of biomolecules, thenonconductive layer can serve as a capture layer for the binder, targetprotein, the surrogate target, or the affinity ligand. For example, onan electrode designed to detect human chorionic gonadotropin (hCG), thenonconductive layer could be an anti-hCG capture antibody, on anelectrode designed to detect a ligand, a receptor molecule could serveas the nonconductive layer. Alternatively, the nonconductive layer canbe a biomolecule that binds the capture molecule such as protein A for acapture antibody or an antibody directed against the capture molecule(i.e. an anti-streptavidin antibody for a binding assay usingstreptavidin as the capture molecule or an anti-receptor antibody for areceptor-based assay). Regardless of the nature of the nonconductivelayer, this layer will ultimately be placed in contact with a solutioncontaining the mediator prior to electrochemical detection.

[0095] Generally, a reference electrode and an auxiliary electrode arealso placed in contact with the mediator solution in conjunction withthe detection electrode. Suitable reference electrodes are known in theart and include, for example, silver/silver chloride (Ag/AgCl)electrodes, saturated calomel electrodes (SCE), and silver pseudoreference electrodes. A suitable auxiliary electrode is a platinumelectrode.

[0096] The detection of the electrochemical signal produced by thecatalytic oxidation-reduction of labels permits the determination of thepresence or absence of specific substances in a sample. As used herein,terms such as determining or detecting “the presence or absence” of asubstance as used to describe the instant invention, also includequantitation of the amount of the substance. In the invention, thetransition metal mediator is oxidized by an electrode. Then, themediator is reduced by the label and then reoxidized at the electrode.Thus, there is electron transfer from the label to the transition metalmediator resulting in regeneration of the reduced form of the transitionmetal mediator as part of a catalytic cycle. The step of determining thepresence or absence of target in a sample typically includes: (i)measuring the electrochemical signal generated by theoxidation-reduction reaction of the mediator at electrodes that are andare not capable of specifically binding the target, (ii) comparing themeasured signal from the transition metal complex at both electrodes,and then (iii) determining whether or not the electrochemical signalgenerated from the mediator at the electrode that is capable of bindingthe target is essentially the same as, greater than, or less than, theelectrochemical signal generated from the mediator at the electrode thatdoes not bind the target. The step of measuring the electrochemicalsignal may be carried out by any suitable means. For example, thedifference in electrochemical signal may be determined by comparing theelectrochemical signal (such as current or charge) from electrodes whichare and are not capable of binding the target at the same scan rate,mediator concentration, buffer condition, temperature, and/orelectrochemical method.

[0097] The electrochemical signal associated with theoxidation-reduction reaction may be measured by providing a suitableapparatus in electronic communication with the detection electrode. Asuitable apparatus is a potentiostat capable of measuring the electronicsignal that is generated so as to provide an indication of whether ornot a reaction has occurred between the label and the mediator. Theelectronic signal may be characteristic of any electrochemical method,including cyclic voltammetry, normal pulse voltammetry,chronoamperometry, and square-wave voltammetry, with chronoamperometryand cyclic voltammetry being the currently preferred forms.

[0098] In cyclic voltammetry, the potential of the electrochemicalsystem is varied linearly from an initial potential between 0-800 mV toa final potential between 500-1600 mV at a constant scan rate (0.01 mV/sto 200 V/s). When the final potential is reached, the scan direction isreversed and the same potential range is swept again in the oppositedirection. The preferred scan rate for Ru(bpy)₃ ²⁺ is 1-20 V/s with a 0mV initial potential and a 1400 mV final potential. The current iscollected at each potential and the data is plotted as a current versuspotential scan. For lower-potential mediators, such as Os(bpy)₃ ²⁺ andOs(Me₂-bpy)₃ ²⁺, instead of scanning from between 0-800 mV to between500-1600 mV, it is preferable to scan from about between 0-100 mV tobetween 300-1000 mV (vs. a Ag/AgCl reference electrode) because of thelower redox potentials required to oxidize these mediators.

[0099] In chronoamperometry as used in the invention herein, theelectrochemical system is stepped from an initial potential between 0mV-800 mV directly to a final potential between 500-1600 mV and heldthere for some specified period of time (50 μs to 10 s) and the currentis collected as a function of time. If desired, the potential can bestepped back to the initial potential, and the current can be collectedat the initial potential as a function of time. The preferred potentialstep for Ru(bpy)₃ ²⁺ is from between 0-800 mV to 1300 mV.(vs. Ag/AgCl)with a collection time of from 50-1000 ms. For lower potentialmediators, such as Os(bpy)₃ ²⁺ and Os(Me₂-bpy)₃ ²⁺, it is preferable tostep from about 0-100 mV to 300-1000 mV (vs. Ag/AgCl).

[0100] In chronocoulometry, a potential step is also applied. For use inthe invention herein, starting at the initial potential (0 mV-800 mV),the electrochemical system is stepped directly to the final potential(500 mV-1600 mV). The electrochemical system is held at the finalpotential for some specified period of time (50 μs to 10 s) and thecharge is collected as a function of time. Although not presently done,if desired, the potential can be stepped back to the initial potentialand the charge can be collected at the initial potential as a functionof time.

[0101] The typical apparatus that would be used for the inventionherein, may, for example, include a sample container for holding a fluidsample; an electrode, as described S above; and a potentiostat inelectronic communication with the electrode surface. In addition, theapparatus preferably comprises a first member of a binding pair, such asa capture antibody, attached to the electrode or to a nonconductivelayer on the electrode surface. The invention may be used with amicroelectronic device comprising a microelectronic substrate havingfirst and second opposing faces, a conductive electrode on the firstface, and an immobilized binder for the target substance on the secondface sufficiently close to the first face to permit detection of anoxidation-reduction reaction on the second face. The oxidation-reductionreaction assay format may be in either: 1) a sandwich format wherein atarget substance, captured by the immobilized first binder, is detectedby a second labeled binder for the target substance, 2) a direct formatwherein the target substance is captured by the immobilized first binderand is detected directly through labels bound to the target, 3) acompetitive format using a labeled target or labeled surrogate targetwhich competes with the target substance in the sample for binding tothe immobilized binder, 4) a competitive format using a labeled binderand immobilized target substance with which the target substance in thesample competes for binding of the labeled binder, or 5) a binding assayformat using an immobilized first binder, a second labeled binder, and atest sample which may or may not affect the interaction between the twobinders.

[0102] D. Quantitating Target Binding.

[0103] The herein-described method is particularly well-suited to thequantitative detection of protein targets and other binding substances.In the case described in this section, the rate constant for oxidationof labels associated with the bound target by the mediator can bedetermined from the cyclic voltammogram by digital simulation. Undermost conditions, this reaction will obey second-order kinetics, so therate=k[mediator][label] where k is the rate constant that is specificfor the particular label, [mediator] is the concentration of themediator, and [label] is the concentration of label. If k and [mediator]are known, then the quantity of the label, and thus of the target, canbe determined. In practice, a calibration curve for the currentenhancements obtained with different quantities of standard solutionscontaining label is constructed so that the electrochemical signalenhancement observed for an electrode treated with a test sample can beused to obtain directly the quantity of label (and target) bound to theelectrode. This quantity is then related directly to the quantity oftarget present in the test sample.

[0104] E. Labels

[0105] The labels utilized in the invention are selected from the groupconsisting of preselected peptides and preselected nucleotide bases, andmay be endogenous or exogenous labels. The labels do not includetransition metal complexes, which are used in the invention as mediatorsto transfer electrons to the conductive substrate. The labels have anoxidation potential approximately equal to or less than that of thetransition metal mediator.

[0106] 1. Endogenous

[0107] The method of the invention may be used to electrochemicallydetect targets containing endogenous labels, for example, particularamino acids in proteins. Endogenous labels are moieties that arecontained naturally within any of the binding members of the assay. Forthe purposes of electrochemical protein detection, endogenous labels areoxidized or reduced in a catalytic reaction with a mediator. In theprotein-detection system, these moieties include amino acids that areoxidized by catalytic mediated electrochemistry in the potential rangeof interest (600-1200 mV) and at potentials below that required for theoxidation of water. This includes cysteine, tyrosine, tryptophan, andhistidine. Other amino acids are also oxidizable but not under the assayconditions described here.

[0108] Because amino acids oxidizable in the potential range of 600-1200mV are present in most protein molecules (and hence in targetmolecules), proteins can be directly detected by catalytic mediatedelectrochemistry. This is particularly true for large proteins andproteins rich in tryptophan or tyrosine.

[0109] 2. Exogenous

[0110] Exogenous labels are moieties that are added to binding membersor targets by synthetic, artificial, natural, or other means. The roleof exogenous labels is to impart electrochemical activity on a moleculethat would otherwise be electrochemically inactive or to increase theelectrochemical activity of an already active molecule. Examples ofexogenous labels used for mediated catalytic electrochemical detectioninclude peptides, peptides with modified amino acids, otherproteinaceous electron donor and acceptor compounds, andoligonucleotides containing preselected nucleotide bases that undergooxidation-reduction by mediated electrochemistry. Other electron donoror acceptor compounds that can be covalently attached to proteins may beused as labels for electrochemical detection of protein targets andother substances and would be obvious to those skilled in the art. Inparticular, donor compounds that are oxidized at potentialsapproximately ≦0.6 V (vs. Ag/AgCl) are useful as labels because they canbe oxidized by mediated electrochemistry under conditions in which thereis no background signal from oxidation of nucleic acids or amino acidspresent in the assay. Examples of low-potential labels are peptidescontaining the modified amino acids 5-hydroxytryptophan (Examples 1, 6,and 7); 3-aminotyrosine; and 3,4-dihydroxyphenylalanine. These modifiedamino acids each have an oxidation potential approximately ≦0.47 V (vs.Ag/AgCl) and are well-suited to react in a mediated catalyticoxidation-reduction reaction with the transition metal mediator,Os(Me₂-bpy)₃ ²⁺, which has an oxidation-reduction potential of about0.47 V (vs. Ag/AgCl).

[0111] A number of labels that have been previously described fordetection of binding interactions are not well-suited for use herein andare not included in this application. For example, omitted as labels formediated electrochemical detection are transition metal complexes andenzyme labels that require a substrate to generate electrochemical oroptical signal through enzymatic catalysis. In the mediated catalyticelectrochemical detection of the invention, the transition metal complexacts as a catalyst and not as a label.

[0112] F. Assay Formats

[0113] The general method of detection of binding interactions of theinstant invention as described above can take any one of severalformats, including conventional sandwich assays, competitive assays, orassays with direct target detection. These assays may be based onimmunological affinity or on affinities that are based onreceptor-ligand, protein-protein, or DNA-protein interactions. Cellreceptors for proteins which can be used in the instant invention asbinders include but are not limited to receptors for transport proteins(i.e., transferrin receptor) (Testa, U., et al., Crit. Rev. Oncog.,1993, 4, 241), receptors for hormone/growth factors (i.e., epidermalgrowth factor, insulin, nerve growth factor) (Ullrich, A. et al., Cell,1990, 61 203; Baxter, R. C., Am. J. Physiol. Endocrinol. Metabol, 2000,278, E967), and G-protein coupled receptors for hormones such asluteinizing hormone, follicle-stimulating hormone, and thyroidstimulating hormone (Schoneberg, T., et at., Mol. Cell Endocrinolo.,1999, 151 181). Receptors of bacterial origin (Modun, B. J., et al.,Microbiology, 1998, 144-1005; Schryvers, A. B., et al., Adv. Exp. Med.Biol., 1998, 443 123) and viral origin (Bella, J., et al., J. Struct.Biol, 1999, 128 69; Domingo, E., et al., Virus Res., 1999, 62 169) mayalso be used in the instant invention. Extracellular matrix proteins(ECM) can be used to detect ECM-binding proteins (Najjam, S., et al.,Cytokine, 1997, 9 1013). DNA can be immobilized as a binding member forDNA-binding proteins such as transcription factors (activators,repressors, or regulators) (McGown, L. B., et al., Anal. Chem., 1995, 67663A). Mediated electrochemical detection of binding interactions mayalso be utilized to evaluate drug candidates for their effects onprotein-protein and other biological interactions. As such, thetechnology described here provides a versatile binding assay for drugdiscovery which can be applied to a variety of drug target-druginteractions. As used herein the term “target protein” includesproteins, glycoproteins, lipoproteins, protein fragments, polypeptides,glycoprotein fragments and lipoprotein fragments.

[0114] 1. Sandwich

[0115] Briefly, in the sandwich assay format, the procedure consists ofmodifying the electrode with the first member of the binding pair (i.e.,antibody, receptor, or DNA), adding the sample, which may or may notcontain the target protein or target substance, then adding the secondbinding member, washing to remove unbound reagents, and adding mediator.Electrochemical interrogation is performed, and enhanced cyclicvoltammetry or chronoamperometry signal relative to a control indicatesthe presence of the target protein or target substance in the sample.

[0116] In this format, the target in the sample is detected via captureby a solid-phase immobilized first binder, such as an antibody, antibodyfragment, receptor protein or DNA to form a target complex, followed bythe binding of the captured target by a labeled second binder to form a3-member target complex. In a preferred embodiment, the second bindercontains only endogenous labels (i.e., electrochemically active aminoacids) and the presence of target in a sample is evident from theincreased current generated by the target complex. In contrast,significantly less current is generated with samples not containing thetarget since complex formation does not occur, and thus, current isgenerated only by any endogenous label in the solid phase immobilizedbinder alone.

[0117] In a second preferred embodiment of the sandwich assay, thecurrent generated by the first preferred embodiment is enhanced by theaddition of a third binder that recognizes the second binder on thetarget complex to create a 4-member complex. This is analogous to theuse of secondary binders in classical immunoassays. The preferredmediator for the first two embodiments (above) is Ru(bpy)₃ ⁺² which hasa potential of about 1.05 V or Os(bpy)₃ ⁺² which has a potential of 0.65V (vs. Ag/AgCl).

[0118] In a third preferred embodiment of the sandwich assay format, thesecond or third binder is covalently labeled with labels such asoligonucleotides, proteins, peptides, or peptides containing modifiedamino acids with lower redox potentials (approximately ≦0.6 V vs. AAgCl),. Mediators matched to these lower potentials, such asOs(Me₂-bpy)₃ ²⁺, are used with the low potential labels. In addition,the second or third binder may be labeled with certain electron donorcompounds that also have low potentials.

[0119] In the instant invention, an alternative to the above sequencesteps for the method of detection is to mix the sample with the secondbinder prior to exposure of the mixture to the immobilized first binder,such that the binding of the second binder occurs prior to binding ofthe target to the immobilized first binder.

[0120] 2. Competitive

[0121] In the competitive assay format, the target competes with alabeled target for binding to an immobilized binder. For example, thebeta chain of the hormone, human chorionic gonadotropin (hCG), can belabeled with a peptide rich in tyrosine or an oligonucleotide containingguanine and shown to bind to rabbit antibody specific for the beta chainof hCG. The detection of hCG in a sample is possible by the competitionof the hCG with the labeled beta chain for the beta chain-specificantibody. In this scenario, the electrochemical signal is high in theabsence of target hCG, and the electrochemical signal decreases iftarget hCG competes with the labeled beta-chain for the immobilized hCGbeta chain-specific antibody. In a similar manner, a labeled surrogatetarget bound to an immobilized binder may be displaced by target presentin a test sample, resulting in a decrease in electrochemical signal. Thecompetitive format is particularly suitable for detecting bindinginteractions of small molecules such as drugs, steroids and vitamins(Example 7).

[0122] 3. Direct

[0123] In the direct target detection assay, the steps are the same asfor the sandwich assay except a labeled second binder is not added. Thelabeled second binder is not required in this case because the targetprotein has the property of being electrochemically active itself, andallows direct mediated electrochemical detection of the target. Thisapproach can be used particularly for large proteins (i.e., ≧150 kD),such as antibodies or other globulins that contain many amino acids andthus are able to generate a significant electrochemical current bythemselves through a catalytic oxidation-reduction reaction with amediator such as Ru(bpy)₃₊₂.

[0124] 4. Competitive Assay for Immobilized Target Substance

[0125] In this format, a target substance or surrogate target substanceis immobilized on the electrode surface and exposed to the sample (whichmay or may not contain the target substance) and a labeled binder(either endogenous or exogenous). As is normally used in this art, forexample, in drug discovery, the surrogate target substance has a lowerbinding affinity than the target substance for the labeled binder. Inthis embodiment of the invention, the electrochemical signal is high inthe absence of the target substance in the sample due to the binding ofthe labeled binder to the immobilized surrogate target substance, andthe electrochemical signal decreases if target substance present in thesample competes with the immobilized surrogate target substance forbinding of the labeled binder.

[0126] 5. Binding Interaction Assay

[0127] In this format, a first binder that is a member of a binding pairis immobilized on the electrode surface. The immobilized binder isexposed to a test sample and to a second binder that is a member of thebinding pair in order to determine the effect of the test sample on thebinding interaction between the first and second binders. The testsample may comprise a substance that facilitates, inhibits, or does notaffect binding of the two binders. For example, the test sample couldcontain a drug candidate that prevents two proteins from binding to eachother, or the test sample could contain a drug candidate that enhancesthe binding interaction. Thus, this assay format can be used to screenpotential drug compounds in order to determine the effect they have on abinding interaction. The mode of action by which the test sample affectsthe binding interaction includes but is not limited to blocking orenhancing the binding of one of the binders and inducing aconformational change in the binding site. In contrast to the aboveassay formats where the intention is to detect the presence or absenceof a substance using catalytic mediated electrochemistry, the bindinginteraction assay format is designed to detect the effect of a substanceon a binding interaction between members of a binding pair usingcatalytic mediated electrochemistry.

[0128] The features of the present invention will be more clearlyunderstood by reference to the following examples, which are not to beconstrued as limiting the invention.

EXAMPLES Example 1 Electrochemical Detection of Tyrosine, Tryptophan and5-Hydroxytryptophan in Solution Using Mediated Catalytic Oxidation

[0129] The presence of tyrosine, tryptophan and 5-hydroxytryptophan wasdetermined in a solution using cyclic voltammetry at a scan rate of 300mV/s (FIG. 1A-1C). The working, reference and counter electrodes wereITO, Ag/AgCl, and platinum wire, respectively. The substrate (aminoacid) concentration was 100 μM, and the mediator concentration was 20 μMwith 50 mM sodium phosphate, pH 7.5 as the supporting electrolyte. Themediators used were A) Ru(bpy)₃ ²⁺, B) Os(bpy)₃ ²⁺, and C) Os(Me₂-bpy)₃²⁺. A dramatic increase in the anodic (oxidative) current from themediator in the presence of substrate provides a quantitative measure ofthe amount of substrate in solution based on the catalytic currentenhancement due to reaction between the oxidized mediator and thesubstrate. The selective reactivity of the different mediators with thesubstrates (i.e., only 5-hydroxytryptophan is detected by Os(Me₂-bpy)₃²⁺, both 5-hydroxytryptophan and tyrosine are detected by Os(bpy)₃ ²⁺,and 5-hydroxytryptophan, tyrosine, and tryptophan are detected byRu(bpy)₃ ²⁺) demonstrates the selectivity of the mediatedelectrochemical detection technique.

Example 2 Attachment of an Exogenous Electrochemical Label to a SignalMolecule

[0130] Electrochemical labels that contain a primary amine can beattached to other molecules that contain primary amines. An example ofthis method is the coupling of a peptide or oligonucleotide modifiedwith an alkyl amine linker to lysine residues on an antibody using theheterobifunctional crosslinkers, N-succinimidyl-S-acetylthioacetate(SATA) andsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC)(both from Pierce Chemical Co., Rockford, Ill.). Both of thecrosslinkers contain an amino-reactive N-hydroxysuccinimide (NHS) ester;however, SATA possesses a protected thiol, while SMCC has athiol-reactive maleimide functionality. Thus, once the label is modifiedwith SATA and the antibody is modified with SMCC, the label and antibodycan be covalently coupled under conditions in which the protected SATAthiol is liberated and reacts with the maleimide group of SMCC. Atypical coupling procedure is described in detail in the instructionprovided by the manufacturer with these reagents. In the presentapplication, a label containing one primary amine (such as a peptide oroligonucleotide with an amino terminus) and an antibody are separatelyreacted with a 20-fold excess of SATA and sulfo-SMCC, respectively, for2 hr at room temperature. Second, excess coupling reagent is removedfrom each reaction by gel filtration chromatography with G-25 Sephadexresin (Sigma, St. Louis, Mo.). Typically, the ratio of crosslinkerincorporation is 1:1 for the label and 3-8:1 for the antibody. Third,the modified label and antibody are then reacted at a ratio of 10-20SATA per SMCC for 2 hr at room temperature in the presence of 50 mMhydroxylamine. Finally, the labeled antibody is separated from theexcess free label by gel filtration or successive washes in acentrifugal concentration device.

Example 3 Detection of Passively Bound Protein (IgG) with Two SolubleBinders—The First Binder Being Specific for the IgG and the SecondBinder Being Specific for the First Binder

[0131] Mouse IgG (mIgG) and human IgG (IgG) were passively adsorbed toITO overnight as follows: Fifty microliters of a 50 μg/ml IgG solutionin 50 mM sodium acetate buffer, pH 5, was applied to 0.28 cm² areas ofITO delineated in a 96-well microtiter plate format. The ITO microtiterwells were formed by fusing a piece of ITO coated glass to the bottom ofa 96-well microtiter plate upper portion. Protein adsorption was allowedto proceed for 15 hr. This was followed by treatment of ITO microtiterwells with 200 μl of 0.1% (w/v) 80% hydrolyzed polyvinyl alcohol (PVA)in 50 mM sodium acetate, pH 5.8 for 2 hr at room temperature to blockany unreacted binding sites on the ITO surface and on the polystyrenemicrotiter plate upper portion. After washing with PBS, the wells weretreated for 2 hr with the first binder (goat anti-mouse IgG, 15 μg/ml,100 μl/well)(Sigma Chemical). The goat anti-mouse IgG antibody had beenaffinity isolated on a mIgG column and adsorbed with human IgG to removeany reactivity specific for hIgG. After washing the ITO with PBS, thewells were treated for 2 hr with the second binder (rabbit anti-goatIgG, 15 μg/ml)(Sigma Chemical). Washed wells were subsequently evaluatedusing cyclic voltammetry (2.5 V/sec) with 50 μM Ru(bpy)₃ as redoxmediator (FIGS. 2A-2B). As shown in FIG. 2A, the addition of the firstbinder to ITO with adsorbed mIgG resulted in a significant currentincrease over that seen with adsorbed mIgG alone (FIG. 2A #3 versus #2),indicating that the first binder was binding the mIgG. A further currentincrease was observed when incubation with the first binder was followedby incubation with the second binder (FIG. 2A #4 versus #3. None ofthese increases was observed when the same antibody combinations wereapplied to ITO adsorbed with hIgG (FIG. 2B).

Example 4 Electrochemical Detection of Human Chorionic Gonadotropin(hCG) Antigen on an ITO Electrode in a Sandwich Immunoassay Using aSignal Antibody Labeled with an Exogenous Oligonucleotide Label

[0132] Electrochemical detection of hCG captured on the surface of anITO electrode was demonstrated using ITO modified with a rabbitanti-beta chain hCG capture antibody (Biostride, Inc., Redwood City,Calif.)(FIG. 3). Capture antibody was passively adsorbed onto ITOovernight from a solution of 20 μg/ml antibody in 50 M sodium acetate,pH 5.0. The ITO was then blocked with 0.1% (w/v) 80% hydrolyzedpolyvinyl alcohol (PVA) in 50 mM sodium acetate, pH 5.8 for 2 hr at roomtemperature to reduce non-specific binding of the antigen and signalantibody. After blocking, the ITO was washed three times with phosphatebuffered saline (PBS). The PVA-blocked, capture-antibody modified ITOwas then treated with 0, 10, or 50 ng/ml hCG (Sigma) in PBS. After 2 hrincubation, the ITO electrodes were washed three times with PBS andtreated with labeled and unlabeled goat anti-alpha chain hCG antibody(Biostride, Inc.) at 25 μg/ml in PBS. The label was a nineguanine-containing oligonucleotide and was present at a stoichiometry offive labels per signal IgG molecule. The ITO electrodes were then usedto collect cyclic voltammograms in the presence of 50 μM Ru(bpy)₃ ²⁺ in50 mM phosphate buffer, pH 7.0. The increased electrochemical signalfrom ITO exposed to hCG and the second labeled antibody (FIG. 3, #4 and#5) relative to ITO that was exposed to no hCG (FIG. 3, #2) indicatesthe specific electrochemical detection of hCG. The increased signal fromlabeled (FIG. 3, #4 and #5) versus unlabeled (FIG. 3, #3) secondantibody in the presence of hCG reflects the signal enhancement obtainedfrom the exogenous electrochemical label.

Example 5 Electrochemical Detection of Human Chorionic Gonadotropin(hCG) Antigen on an ITO Electrode in a Sandwich Immunoassay Using aSignal Antibody Labeled with an Exogenous Peptide Label

[0133] Electrochemical detection of hCG captured on the surface of anITO electrode was demonstrated using ITO modified with a rabbitanti-beta chain hCG capture antibody (Biostride, Inc., Redwood City,Calif.)(FIG. 4). Capture antibody was passively adsorbed onto ITOovernight from a solution of 20 μg/ml antibody in 50 mM sodium acetate,pH 5.0. The ITO was then blocked with 0.1% (w/v) 80% hydrolyzedpolyvinyl alcohol (PVA) in 50 mM sodium acetate, pH 5.8 for 2 hr at roomtemperature to reduce non-specific binding of the antigen and signalantibody. After blocking, the ITO was washed three times with phosphatebuffered saline (PBS). The PVA-blocked, capture-antibody modified ITOwas then treated with 100 ng/ml hCG (Sigma) in PBS. After 2 hrincubation, the ITO electrodes were washed three times with PBS andtreated with labeled and unlabeled goat anti-alpha chain hCG antibody(Biostride, Inc.) at 30 μg/ml in PBS. The label was a fivetyrosine-containing peptide and was present at a stoichiometry of fivelabels per signal IgG molecule. The ITO electrodes were then used tocollect cyclic voltammograms in the presence of 50 μM Os(bpy)₃ ²⁺ in 50mM phosphate buffer, pH 7.0. The increased electrochemical signal fromITO exposed to hCG and peptide labeled antibody (FIG. 4A, #4) relativeto ITO that was exposed to no hCG (FIG. 4A, #3) indicates the specificelectrochemical detection of hCG. The increased signal from labeled(FIG. 4A. #4) versus unlabeled (FIG. 4B, #4) second antibody reflectsthe signal enhancement from the exogenous label.

Example 6 Electrochemical Detection of an IgG Labeled with an ExogenousPeptide Containing a Modified Low Potential Amino Acid(5-hydroxytryptophan) and Using the Low Potential Mediator,Osmium²⁺(4,4′-dimethyl-2,2′-bipyridine)₃(“Os(Me₂-bpy)₃ ²⁺”)

[0134] The exogenous peptide label was a 21-mer containing three5-hydroxytryptophan residues. The peptide labeled goat IgG (IgG-WOH) wascaptured with an affinity purified rabbit anti-goat IgG (Sigma Chemical)that was passively adsorbed on the ITO surface from a 50 mM sodiumacetate buffer, pH 5. After adsorption (i.e., immobilization) of thecapture IgG, the ITO was blocked with 0.1% (w/v) 80% hydrolyzedpolyvinyl alcohol (PVA) (Sigma Chemical) in 50 mM sodium acetate, pH 5.8for 2 hr at room temperature. ITO with capture IgG was treated with theIgG-WOH at 25 μg/ml for 4 hr (FIG. 5A). The ITO electrode was then usedto collect the cyclic voltammograms in duplicate in the presence of 50μm Os(Me₂-bpy)₃ ²⁺ in 50 mM phosphate buffer, pH 7. As controls, ITOelectrodes with non-immune rabbit IgG as capture were treated withIgG-WOH (FIG. 5B), and ITO electrodes with rabbit anti-goat IgG ascapture were treated with unlabeled goat IgG (FIG. 5C). The increasedelectrochemical signal from ITO with rabbit anti-goat capture IgGexposed to IgG-WOH indicates electron transfer from the modified aminoacid to the Os(Me₂-bpy)₃ ²⁺ mediator (FIG. 5A). Little or no increase inthe electrochemical signal is seen when non-immune rabbit IgG was usedinstead of rabbit anti-goat IgG as capture (FIG. 5B) or when goat IgGwas not labeled with the modified peptide (FIG. 5C). The use of thismodified low potential amino acid as a label with the low potentialmediator, Os(Me₂-bpy)₃ ²⁺, permits protein detection with very lowbackground signal from endogenous amino acids in the assay reagents ortarget proteins.

Example 7 Detection of Biotin (Vitamin H) in a Competitive Assay UsingLabeled Biotin

[0135] Electrochemical detection of biotin is demonstrated using ITOmodified with neutravidin (Pierce Chemical Co., Rockford, Ill.) andbiotin labeled with a peptide containing 5-hydroxytryptophan (FIG. 6).Neutravidin was passively adsorbed onto ITO overnight, from a solutionof 20 μg/ml neutravidin in 50 mM sodium acetate, pH 5.0. The ITO wasthen blocked with 0.1% (w/v) 80% hydrolyzed polyvinyl alcohol (PVA)(Sigma Chemical) in 50 mM sodium acetate, pH 5.8 for 2 hr at roomtemperature to reduce non-specific binding. After blocking, the ITO waswashed three times with phosphate buffered saline (PBS). ThePVA-blocked, neutravidin-modified ITO was then treated with PBS only(untreated), 1 μM labeled biotin in PBS, or 1 μM labeled biotin plus 160μM unlabeled biotin in PBS. After 2 hr incubation, the ITO electrodeswere washed with PBS, blotted dry, and used to collect cyclicvoltammograms (2.5 V/s) in the presence of 50 μM Os(Me₂-bpy)₃ ²⁺ in 50mM phosphate buffer, pH 7.3. The results show a decrease inelectrochemical signal from the sample containing unlabeled biotinindicating a decrease in the amount of labeled biotin bound on thesurface of the ITO electrode due to the presence of unlabeled biotin inthe sample. The 21-mer peptide label contained three 5-hydroxytryptophanresidues.

[0136] While the invention has been described with reference to specificembodiments, it will be appreciated that numerous variations,modifications, and embodiments are possible, and accordingly, all suchvariations, modifications, and embodiments are to be regarded as beingwithin the spirit and scope of the invention.

1-112. (Canceled)
 113. A labeled member of a binding pair useful formediated catalytic electrochemistry, comprising: a) a binder selectedfrom the group consisting of proteins, protein fragments, recombinantproteins, recombinant protein fragments, extracellular matrix proteins,ligands, carbohydrates, steroids, hormones, drugs, drug candidates,immunoglobulins, receptors of eukaryotic, prokaryotic or viral origin,and oligonucleotides; and b) an exogenous peptide label containing oneor more modified amino acids capable of being oxidized in anoxidation-reduction reaction at potentials below those of naturallyoccurring amino acids.
 114. The labeled member of a binding pair ofclaim 113, wherein the binder is an antibody.
 115. The labeled member ofa binding pair of claim 113, wherein there are at least two modifiedamino acids in the peptide label.
 116. The labeled member of a bindingpair of claim 113, wherein the modified amino acids in the peptide labelare selected from the group consisting of derivatives of tyrosine andderivatives of tryptophan.
 117. The labeled member of a binding pair ofclaim 116, wherein the modified amino acids in the peptide label areselected from the group consisting of 5-hydroxytryptophan;3-aminotyrosine; and 3,4-dihydroxyphenylalanine.